The overall hypothesis of this proposal is that neuronal Ca++ homeostasis plays a major role in opioid analgesia and tolerance by affecting signal transduction systems and K+ channel regulation, which ultimately translates into longterm change in endogenous opioid and neurokinin peptide release. The aims of this proposal are to: (1) understand how opioid analgesia and tolerance, and (3) elucidate the role of endogenous opioids and neurokinins in opioid analgesia and tolerance. Regarding Ca++- sensitive systems (1), manipulations intended to increase cytosolic Ca++ block acute opioid analgesia, while Ca++ channel antagonists potentiate analgesia. Alternatively, a disruption in Ca++ homeostasis accompanies the development of morphine tolerance, as evidenced by increases in synaptosomal 45 Ca++ uptake and higher free intracellular Ca++ levels in brain and spinal cored synaptosomes. We propose to test the hypothesis that opioid analgesia and tolerance are modulated by the relative activity of calmodulin and the subsequent activation of calmodulin-sensitive adenylate cyclase (and indirectly PKA activity), and/or multifunctional calmodulin-dependent protein kinase II (CaM kinase II). Another potential contributor to analgesia and tolerance is related to the observation that stimulation- induced increases in cytosolic Ca++ activates phospholipase C (PLC) independent of receptor G-protein coupling. We will test the hypothesis that the phosphatidylinositol system plays a role in opioid analgesia, and that opioid tolerance-induced disruptions in Ca++ homeostasis are related to PLC and the subsequent production of IP3 and activation of protein kinase C (PKC). Regarding K+ channel regulation in analgesia and tolerance (2), we have clearly demonstrated that acute morphine analgesia is antagonized by blockers of potassium channels such as glyburide and apamin. Consistent with this are in vitro observations that K+ channel antagonists prevent mu opioid-stimulated k+ efflux and neuronal membrane hyperpolarization. We will test the hypothesis that chronic administration of mu opioids decreases potassium efflux from neurons as compensatory mechanism in the process of the development of tolerance. Regarding neurokinins and endogenous opioids (3), we will evaluate their roles in analgesia and in the development and regulation of opioid tolerance. We hypothesize that besides playing a modulatory role on pain pathways stimulated by excitatory amino acid neurotransmitters, neurokinins mediate endogenous opioid release. We and others have demonstrated that neurokinin agonists produce a naloxone-sensitive analgesia which we propose results from endogenous opioid release. We will test the hypothesis that opioid tolerance involves changes in spinal neurokinin levels or release, and the neurokinins participate in opioid analgesia by releasing endogenous opioids and altering Ca++ homeostasis. These studies are intended to clarify the role of neurokinins and endogenous opioids in tolerance, since their participation in tolerance is frequently obscured.